PROJECT SUMMARY/ABSTRACT This project will focus on understanding mechanisms of intestinal epithelial cell (IEC) transformation. We define early features of transformation as the augmentation of proliferation, stem cell fate, and repression of IEC differentiation. Here, we seek to understand the role of a specific transcription factor (PLAGL2) we have recently identified as a target repressed by Let-7 microRNAs as a part of a recently published manuscript in Stem Cell Reports, showing that PLAGL2 drives stem cell fate. Our previous studies indicate that Let-7 is a potent repressor of tumorigenesis and intestinal stem cell fate. Many Let-7 targets have been implicated as oncogenes in multiple tissues, including MYC and RAS, but in our models of Let-7 manipulation in the intestine, we do not find significant effects on these targets. We present evidence that our newly identified Let- 7 target, PLAGL2, is commonly up-regulated (>60%) in pre-cancerous and cancerous colorectal tumors in humans, with expression inversely proportional to Let-7 miRNAs. This potentially represents a major pathway of tumorigenesis as both Let-7 and target mRNAs are deregulated in the majority of colorectal cancers (CRC), and is thus highly relevant. In human CRC cell lines we find that PLAGL2 is necessary for driving a malignant phenotype, while in normal IEC organoids PLAGL2 can drive early features of transformation. First, to identify roles for PLAGL2 in IEC transformation we will look at the potential for this factor to drive tumorigenesis through targeted over-expression in the intestine, in the context of Let-7 depletion through LIN28B over- expression. Using a newly generated floxed allele, we will also determine whether PLAGL2 is required for driving early stages of IEC transformation in this model. Second, we will use our novel CRISPR somatic mutagenesis model of intestinal tumorigenesis to examine interaction and cooperation of PLAGL2 with canonical CRC oncogenic pathways. This model randomly mutates the tumor suppressors Apc, Pten, Smad4, and Trp53 to generate adenomas and adenocarcinomas, which allows us to study PLAGL2 across an array of malignant stages, tumor types, in the context of different, but defined, mutations. Third, we will explore the roles of relevant PLAGL2 transcriptional targets, including target genes we have already identified, in driving early processes of transformation. Through new assays and reporters in organoids, we will also characterize Wnt signaling activity in the clonogenic cell of origin that is mobilized by aberrant PLAGL2 expression. These investigations will likely illuminate how this newly discovered Let-7 target, PLAGL2, drives pre-malignant changes and tumorigenesis. Considering its widespread up-regulation in pre-cancerous and cancerous growths in the intestine, PLAGL2 may represent a keystone target for therapeutic inhibition. Additionally, given the potent effects on stem cell fate that we see driven by PLAGL2, this factor may also play a key role in the early definition, or later maintenance, of intestinal stem cells.